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FoxP3(+) confers suppressive properties and is confined to regulatory T cells (T(reg)) that potently inhibit autoreactive immune responses. In the transplant setting, natural CD4(+) T(reg) are critical in controlling alloreactivity and the establishment of tolerance. We now identify an important CD8(+) population of FoxP3(+) T(reg) that convert from CD8(+) conventional donor T cells after allogeneic but not syngeneic bone marrow transplantation. These CD8(+) T(reg) undergo conversion in the mesenteric lymph nodes under the influence of recipient dendritic cells and TGF-β. Importantly, this population is as important for protection from GVHD as the well-studied natural CD4(+)FoxP3(+) population and is more potent in exerting class I-restricted and antigen-specific suppression in vitro and in vivo. Critically, CD8(+)FoxP3(+) T(reg) are exquisitely sensitive to inhibition by cyclosporine but can be massively and specifically expanded in vivo to prevent GVHD by coadministering rapamycin and IL-2 antibody complexes. CD8(+)FoxP3(+) T(reg) thus represent a new regulatory population with considerable potential to preferentially subvert MHC class I-restricted T-cell responses after bone marrow transplantation.

The presentation pathways by which allogeneic peptides induce graft-versus-host disease (GVHD) are unclear. We developed a bone marrow transplant (BMT) system in mice whereby presentation of a processed recipient peptide within major histocompatibility complex (MHC) class II molecules could be spatially and temporally quantified. Whereas donor antigen presenting cells (APCs) could induce lethal acute GVHD via MHC class II, recipient APCs were 100-1,000 times more potent in this regard. After myeloablative irradiation, T cell activation and memory differentiation occurred in lymphoid organs independently of alloantigen. Unexpectedly, professional hematopoietic-derived recipient APCs within lymphoid organs had only a limited capacity to induce GVHD, and dendritic cells were not required. In contrast, nonhematopoietic recipient APCs within target organs induced universal GVHD mortality and promoted marked alloreactive donor T cell expansion within the gastrointestinal tract and inflammatory cytokine generation. These data challenge current paradigms, suggesting that experimental lethal acute GVHD can be induced by nonhematopoietic recipient APCs.

Although the effects of type II-IFN (IFN-γ) on GVHD and leukemia relapse are well studied, the effects of type I-interferon (type I-IFN, IFN-α/β) remain unclear. We investigated this using type I-IFN receptor-deficient mice and exogenous IFN-α administration in established models of GVHD and GVL. Type I-IFN signaling in host tissue prevented severe colon-targeted GVHD in CD4-dependent models of GVHD directed toward either major histocompatibility antigens or multiple minor histocompatibility antigens. This protection was the result of suppression of donor CD4(+) T-cell proliferation and differentiation. Studies in chimeric recipients demonstrated this was due to type I-IFN signaling in hematopoietic tissue. Consistent with this finding, administration of IFN-α during conditioning inhibited donor CD4(+) proliferation and differentiation. In contrast, CD8-dependent GVHD and GVL effects were enhanced when type I-IFN signaling was intact in the host or donor, respectively. This finding reflected the ability of type I-IFN to both sensitize host target tissue/leukemia to cell-mediated cytotoxicity and augment donor CTL function. These data confirm that type I-IFN plays an important role in defining the balance of GVHD and GVL responses and suggests that administration of the cytokine after BM transplantation could be studied prospectively in patients at high risk of relapse.

The recent shift to the use of stem cells mobilized by granulocyte colony-stimulating factor (G-CSF) for hematopoietic transplantation has increased chronic graftversus-host disease (GVHD), although the mechanisms of this are unclear. We have found that G-CSF invokes potent type 17 rather than type 1 or type 2 differentiation. The amplification of interleukin-17 (IL-17) production by G-CSF occurs in both CD4 and CD8 conventional T cells and is dependent on, and downstream of, G-CSF-induced IL-21 signaling. Importantly, donor IL-17A controls the infiltration of macrophages into skin and cutaneous fibrosis, manifesting late after transplantation as scleroderma. Interestingly, donor CD8 T cells were the predominant source of IL-17A after transplantation and could mediate scleroderma independently of CD4 T cells. This study provides a logical explanation for the propensity of allogeneic stem cell transplantation to invoke sclerodermatous GVHD and suggests a therapeutic strategy for intervention.

We have quantified the relative contribution of donor antigen-presenting cell populations to alloantigen presentation after bone marrow transplantation (BMT) by using transgenic T cells that can respond to host-derived alloantigen presented within the donor major histocompatibility complex. We also used additional transgenic/knockout donor mice and/or monoclonal antibodies that allowed conditional depletion of conventional dendritic cells (cDCs), plasmacytoid DC (pDCs), macrophages, or B cells. Using these systems, we demonstrate that donor cDCs are the critical population presenting alloantigen after BMT, whereas pDCs and macrophages do not make a significant contribution in isolation. In addition, alloantigen presentation was significantly enhanced in the absence of donor B cells, confirming a regulatory role for these cells early after transplantation. These data have major implications for the design of therapeutic strategies post-BMT, and suggest that cDC depletion and the promotion of B-cell reconstitution may be beneficial tools for the control of alloreactivity.

Granulocyte colony-stimulating factor (G-CSF) is often used to hasten neutrophil recovery after allogeneic bone marrow transplantation (BMT), but the clinical and immunological consequences evoked remain unclear. We examined the effect of G-CSF administration after transplantation in mouse models and found that exposure to either standard G-CSF or pegylated-G-CSF soon after BMT substantially increased graft-versus-host disease (GVHD). This effect was dependent on total body irradiation (TBI) rendering host dendritic cells (DCs) responsive to G-CSF by upregulating their expression of the G-CSF receptor. Stimulation of host DCs by G-CSF subsequently unleashed a cascade of events characterized by donor natural killer T cell (NKT cell) activation, interferon-gamma secretion and CD40-dependent amplification of donor cytotoxic T lymphocyte function during the effector phase of GVHD. Crucially, the detrimental effects of G-CSF were only present when it was administered after TBI conditioning and at a time when residual host antigen presenting cells were still present, perhaps explaining the conflicting and somewhat controversial clinical studies from the large European and North American BMT registries. These data have major implications for the use of G-CSF in disease states where NKT cell activation may have effects on outcome.